Field of the Invention
The present invention relates generally to methods of monitoring binding of a compound to a target macromolecule by increasing the thermal stability of the target macromolecule. The present invention also relates to a novel method of use of enzyme fragment complementation.
Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual compositions or methods used in the present invention may be described in greater detail in the publications and patents discussed below, which may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance or the prior art effect of the patents or publications described.
A number of areas of biology and medicine depend on and develop assays to detect ligand binding to proteins and/or protein fragments. The binding assays help in determining a target which should be safe and druggable. The multibillion dollar pharmaceutical industry depends on these assays to find a drug compound that has an ability to bind to its target protein to perform its function.
A number of protein-ligand interaction assays have been developed and discussed in the past such as labeled and label-free ligand binding assays, structure-based ligand binding assays, thermodynamic ligand binding assays and whole cell ligand binding assays. Further, there is a fluorescence based ligand binding assay wherein a fluorescently labeled ligand binds to a target macromolecule. However, the assay is susceptible to different fluorescence interference and thus leads to undesirable alterations in the binding characteristics of the ligand. A radioactively labeled binding assay is popular for membrane based targets; however, the assay suffers from high cost along with hazards of handling high levels of radioactivity and thus comes with many restrictions for the lab and lab personnel working with the assay. NMR based analysis has also been applied to analyze the detailed structure of proteins and thus to assist in structure based drug design but suffers from a high cost of the assay and a long time required to analyze the spectra.
A previously developed thermal shift assay, also called differential scanning fluorimetry (DSF), is a thermal-denaturation assay that measures the thermal stability of a target protein and a subsequent increase in protein melting temperature upon binding of a ligand to the protein. The thermal stability change is measured by performing a thermal denaturation curve in the presence of a fluorescent dye, such as Sypro Orange. Such methods also involve a step of centrifugation and oil dispensing.
Thus, there is a need for a binding assay that offers a facile, sensitive and precise detection of ligand protein interaction, in a homogeneous assay format.